Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.     

Effect of methylmethane sulfonate-sensitive mutations on nondisjunction and loss of sex chromosomes in Drosophila males

The MMS-sensitive mutants mus(1) 120M1 and mus(1) 121M1 of Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray induced (2000 R) aneuploidy in male germ cells, during different stages of spermatogenesis. In matings of males carrying mus mutation and a doubly marked Y-chromosome (BsYy+) with repair proficient y f females, the frequencies of partial loss, nondisjunctions and especially complete loss were significantly higher than in the control. Apparently, MMS-sensitive mutants deal with meiotic processes and maintenance of chromosome structural stability both in females and in males, in somatic and germ cells.     

Cell-cycle regulation of cohesin stability along fission yeast chromosomes

Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.     

Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.     

Mitotic spatial model: Arrangement of homologous chromosomes

Two different mitotic spatial models (Lavania & Sharma, Biosystems 14: 171–178, 1981: Ashley & Pocock, Genetica 55: 161–169, 1981) for the arrangement of the chromosomes in the nucleus are analysed for the sake of comparison and assessment. In general, the two models propose nearly the same orientation regarding chromosomal organization. However, they differ with respect to the position of homologous chromosomes, suggesting respectively their adjacent or opposite location in somatic arrangement. The two proposals have been analysed in the light of experimental evidence as well as organizational interpretations. The arguments presented here support the adjacent location of somatic homologous chromosomes.     

Saccharomyces cerevisiae linear chromosome stability (lcs) mutants increase the loss rate of artificial and natural linear chromosomes

We isolated mutants of Saccharomyces cerevisiae that lose a 100 kb linear yeast artificial chromosome (YAC) at elevated rates. Mutations in two of these LCS (linear chromosome stability) genes had little or no effect on the loss rate of a circular YAC that had the same centromere and origin of replication as present on the linear YAC. Moreover, mutations in these LCS genes also increased the loss rate of an authentic linear yeast chromosome, chromosome III, but had only small effects on the loss rate of a circular derivative of chromosome III. As these mutants preferentially destabilize linear chromosomes, they may affect chromosome stability through interactions at telomeres. Telomeres are thought to be essential for the protection and complete replication of chromosome ends. The cytological properties of telomeres suggest that these structures may play additional roles in chromosome function. The lengths of the terminal C_1–3A repeats at the ends of yeast chromosomes were unaltered in the linear preferential lcs mutants, suggesting that these mutants do not affect the replication or protection of telomeric DNA. Thus, the linear-preferential lcs mutants may identify a role for telomeres in chromosome stability that is distinct from their function in the replication and protection of chromosomal termini.by J. Huberman     

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.     

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Development of a microarray assay that measures hybridization stoichiometry in moles

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.     

Mitotic chromosomes and the W-sex chromosome of the great horned owl (Bubo V. virginianus)

Summary Mitotic chromosomes from the feather pulp and leucocyte cultures of the great horned owl (Bubo v. virginianus) were analyzed in both the sexes. The largest pair of chromosomes are acrocentrics while those of the second and the third pair have a short arm 1/6th the size of the large one. Chromosomes of the fourth and the fifth pairs have a median constriction while those of the 6th to 9th pairs are all acrocentrics. These are followed in order of size by a large number of smaller chromosomes, the smallest of which appear as dots. In 15 plates out of a total of 21, 82 to 84 chromosomes were counted and it is presumed that either of these numbers represents the diploid number. In the female sex besides the single unpaired 4th Z-chromosome, there is a small metacentric unpaired W chromosome which is nearly equal in size to the chromosomes of the 7th pair.Contribution No. 5729 Scientific Journal Series, Minnesota Agricultural Experiment Station. This work was supported in part by NSF grant GB 2859.     

Mitotic and meiotic chromosomes of Mabuya (Scincidae: Reptilia)

A study was carried out on conventionally stained mitotic and meiotic chromosomes of two species of lizards of the Scincidae: Mabuya caissara and M. macrorhyncha.On the basis of morphological, numerical and metric data, the chromosomes were divided into three groups: two groups of macrochromosomes (A and B) and one of microchromosomes (C). The karyotypes of males and females of both species showed 2n=32 and FN=50 and no differences were detected between the species.     

Characterization of yeast artificial chromosomes containing interleukin genes on human chromosome 5.

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.     

Mitotic chromosome loss induced by methyl benzimidazole-2-yl-carbamate as a rapid mapping method in Saccharomyces cerevisiae.

Mitotic chromosome loss induced by methyl benzimidazole-2-yl-carbamate has been utilized as a rapid and simple method for assigning genes to individual chromosomes in Saccharomyces cerevisiae. This technique relied on the segregation of heterozygous markers in a diploid strain after methyl benzimidazole-2-yl-carbamate treatment due to loss of whole chromosomes. Correlations between the expression of an unmapped gene and that of a previously mapped recessive marker indicated chromosomal linkage. Depending on whether the unmapped gene and the marker were located in coupling or in repulsion, either positive or negative correlations were seen. The chromosomal location of several previously mapped genes were confirmed as a test of the method, and one previously unmapped gene, nib1, was mapped.     

CFU-F from dog marrow: a colony assay and its significance

A colony assay method is described for studying dog fibroblast colony development in marrow cells derived from resected ribs. The assay showed an increased number of fibroblast colony forming units (CFU-F) in cell suspensions prepared from resected ribs compared to cell suspensions prepared from bone marrow aspirates or from peripheral blood. A linear relationship between the number of cells plated and the number of fibroblastoid colonies was demonstrated in each case. The proportion of phagocytic cells was lower in cultures prepared from resected ribs than in those prepared from bone marrow aspirates. Staining for acid phosphatase and with sudan black showed differences between phagocytic cells and non-phagocytic fibroblasts. When left in plastic dishes for 2 hrs, 81% +/- 10% of the CFU-F adhered to the plastic dishes. Velocity sedimentation separation showed a modal sedimentation rate of 6.49 mm/h.     

Mitotic remodeling of the replicon and chromosome structure

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.     

Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.     

Mitotic motors and chromosome segregation: the mechanism of anaphase B

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an 'on/off' switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned 'off', tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.